In Vitro Antibacterial Activity of Citrus limon (L.) Burm against Gentamicin-Resistant Escherichia
coli Complemented with In Silico Molecular Docking of Its Major Phytochemicals
with Ribosome Recycling Factor

Abstract

Objectives: Due to adverse effects of classic antibiotics and accrual emergence of antibiotic-resistant bacteria, seeking alternative therapeutics would be a necessity. This study was first aimed to investigate in vitro antibacterial activity of hydro-alcoholic extracts derived from peels of Citrus limon (L.) Burm (CL) as anecdotally reported in (Kurdish) ethnomedicine. Then, putative mechanisms which may mediate this antibacterial effect were computationally elucidated.

Materials and Methods: Antimicrobial activity was determined against strains E. coli ATTCC 25922. Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentration (MBC) were determined. Molecular docking was performed for major phytocompounds reported in aqueous or hydro-alcoholic extract of peel of CL against Ribosome Recycling Factor (RRF) of E. coli.

Keywords

Introduction

Plants have two sets of metabolites, primary and secondary, which are important for
their life [1]. Secondary metabolite such as sterols, phenolics, steroids, lignins, and tannins
are substances that their presences are not obligatory for growth of plants, but necessary
for protecting against (a)biotic stresses. For instance, secondary metabolites are able to kill
or suppress growth of microbes [2].

Medicinal herbs are a group of plants that could be used for drug development due to the
presence of secondary metabolites, phytochemicals that are beneficial to prevent diseases
and to eliminate pathogenic germs in animals and human [3]. Phytochemicals are able
to prohibit peptidoglycan installation, alter bacterial membrane surface hydrophobicity,
destruct microbial membrane framework, and as well as modulate bacterial quorum
sensing [4].

Antibacterial phytochemicals (phytobiotics) and antibiotics inhibit or kill pathogenic
bacteria via inhibiting proteins or genes that are essential for the life of bacteria. For
example, ribosome recycling is responsible for translating genetic information carried
by mRNAs into specific sequences of amino acids and may increase the efficiency of
translation by recycling ribosomes from one round of translation to another [5]. In this
context, gentamicin is a bactericidal antibiotic that works by irreversibly binding the 30S
subunit of the bacterial ribosome, interrupting protein synthesis, this mechanism of action
is similar to other aminoglycosides, however we encounter to the emergence of gentamicin
resistance bacteria nowadays [6].

and medicinal plant belongs to the Rutaceae family [7]. Its specific
phytochemical screening revealed presence of alkaloids, tannins,
fixed oils, cardiac glycosides, steroids, phenols, flavonoids, carotenoid,
pectin, hesperidin, quercetin, lutein, zeaxanthin, β-cryptoxanthin and
β-carotene [8]. In vitro antibacterial role of peel of CL is mediated
through splitting lipids of bacterial the cell membrane and thus
breaching the cell structure and making it more permeable [9].
In this continuum, aqueous extracts of peel of CL showed highest
antibacterial activity against gram-positive and gram-negative
bacteria including Staphylococcus aureus, Streptococcus pyogenes,
Enterococcus faecalis, Streptococcus pneumonia and Pseudomonas
aeruginosa [10]

Based on (Kurdish) ethnomedicine, this study was aimed
firstly to examine in vitro antibacterial activity of the peel of CL
against Escherichia coli which causes human and animal diseases
and secondly to decipher in silico docking of its major reported
phytochemicals (Figure 1) against ribosome recycling factor as a vital
protein of E. coli.

Materials and Methods

Plant preparation

The lemons were collected from different locations in Iran and Iraq
since March to October 2016. The collected specie was authenticated
by botanist, third author. The peel of CL was air-dried and finely
powdered using a blender. To prepare hydro-alcoholic extract, 10 g of
powdered peels were extracted twice with 100 ml of 70 % ethanol for
48 h at room temperature. The extracted suspensions were filtered
and resulting filtrates were concentrated to complete dryness using
a rotary evaporator and then store at −20 °C until further use. For the
antibacterial activity assays, the extract was dissolved in Dimethyl
Sulfoxide (DMSO) and store at 4 °C as stock solutions.

In vitro antibacterial assay

We used gentamicin to assay the effects of antibiotics on the strains E. coli (ATTCC® 25922TM) at 5 µg.ml-1 of the antibiotics for each well. E. coli was cultured on Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) media. The antibacterial activity of peel of CL on E. coli was measured by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) methods based on broth microdilution method [11]. Serial dilutions of the extract were prepared in a 96-well micro-titer plate. To each well, 100 μl of indicator solution that prepared by dissolving a 10 mg extract in 1 ml of DMSO and 100 μl of MHB were added. Finally, 50 μl of bacterial suspension (106 colony forming unite (CFU).ml-1) were added to each well to achieve a concentration of 104 CFU.ml-1. The plates were wrapped loosely with cling film to ensure that bacteria did not get dehydrated. The plated were prepared in triplicates, and then were incubated at 37°C for 18-24 hours.

In silico antibacterial assay

Simulations of the docking between bacterial target protein
and reported bioactive compounds of CL were successfully
performed using PyRx software (ver. 0.8) and a docking program
(VINA WIZARD) using default parameters as described previously
[12]. For molecular docking, crystal structures of target proteins
were obtained from the Protein Data Bank (PDB) at the Research
Collaboratory for Structural Bioinformatics (http://www.RCSB.org).
The PDB format of target protein have been edited, optimized and
trimmed in Molegro Virtual Docker and Chimera 1.8.1 (http://www.
rbvi.ucsf.edu/chimera) before submission to PyRx. The structures of
the major phytocompounds of CL were retrieved from ZINC database
ver. 12.0 (http://zinc.docking.org/) or DrugBank ver. 5.0 (https://
www.drugbank.ca/; Figure 1).

Figure 1

The phytochemicals that have been reported in the peels of Citrus limon (L.) Burm.

After completion of docking procedure, results had shown as binding affinity (kcal.mol-1) values. More negative the binding affinity means better the orientation of the ligand in the binding site. The selected conformer of ligand has been combined with target protein in Molegro Virtual Docker [13] or Chimera 1.8.1 (http://www.rbvi. ucsf.edu/chimera) and their graphical interface has been analyzed with LigPlot+ software [14].

Results and Discussion

High incidence of nosocomial diseases due to antibiotic
resistance, high prices of synthetic antibiotics and hard access to
prepare synthetic antibiotics in poor countries lead to overuse
of traditional remedies to treat diseases. In this essence, reverse
pharmacognosy help scientists to discover natural bioactive
compounds which are drug or drug-like candidates. Numerous
naturally-occurring components are found in medicinal plant that
owing to their antimicrobial properties could be applied as a valuable
source for antimicrobial medicines [15].

Ligand-Code of ZINC12

Binding affinity (kcal.mol-1)

RMSD/upper bound

RMSD/lower bound

Alpha-carotene-8219947

-6.7

8.50

5.00

Apigenin-11726230

-10.2

2.91

2.05

Beta-carotene-85649478

-10.0

17.60

10.95

Beta-cryptoxanthin-70450846

-8.0

17.92

4.55

D-limonene-967513

-7.6

4.23

1.13

Citral-1529208

-7.5

5.51

2.68

Eriocitrin-33963983

-12.1

24.07

20.67

Alpha-pinene-967579

-7.5

3.19

1.091

Lycopene-8214943

-10.5

2. 15

1.15

Hesperidin-8382286

-13.3

26.99

23.78

Gentamicin-DrugBank

-12.6

3.65

1.65

Note: RMSD: root mean-square deviation is the measure of the average distance between the atoms.

E. coli causes various diseases of gastrointestinal tract in human
and other animals [16]. Strains of E. coli ATTCC 25922 used in this
study was resistant to gentamicin. Gentamicin is a bactericidal,
massively used antibiotic that cause mRNA decoding mistake, prevent
transfer RNA and mRNA translocation, and block ribosome recycling
in bacteria [17]. Our results showed that this strain of E. coli ATTCC
25922 was resistant to gentamicin while sensitive to CL. In sum,
peel of CL has inhibitory at MIC 32 ± 27 (μg.ml-1) and bactericidal
effects at MBC 682 ± 295 (μg.ml-1) on E. coli. In this regard, Fisher
and colleagues studied phytochemicals of the CL peel and their
antimicrobial activities and reported that the essential oil of peel of
CL had good antimicrobial activity against E. coli [18]

In this study, we selected gentamicin as reference antibiotic to
compare its in silico affinity with Ribosome Recycling Factor (RRF;
PDB code 1WIH) with respect to ligands of peel of CL to decipher
which phytocompounds may involve in anti-E coli effects of CL (Table
1 & Figure 2). In this study, we selected RRF as target protein since it
is product of the frr gene in E. coli and are responsible for dissociation
of ribosomes from mRNA after the termination of translation and
recyclizes ribosomes [19]

Based on docking results, gentamicin showed negative binding
affinity with RRF at -12.6 (kcal.mol-1; Table 1) and docked via
hydrophobic interactions and hydrogen bonds (Figure 2). In silico
findings showed that among phytochemicals found in peel of CL,
hesperidin has been docked with lower binding affinity with RRF
with respect to gentamicin

Hesperidin is a flavanone glycoside found in citrus fruits and its
aglycone form is called hesperetin. Hesperidin was first isolated from
albedo of citrus peels [20]. Hesperidin showed the lowest negative
binding affinity against RRF among in silico studied phytochemicals
of CL. It docked with RRF using both hydrophobic interactions and
hydrogen bonds. Hesperidin showed in vivo and in vitro antimicrobial
activities against Aeromonas hydrophila and E. coli, respectively
and its bioactive activities are reviewed previously [21-23]. Its
pharmacological activities promise its potential to be considered as a
drug-like compound.

Eriocitrin, eriodictyol 7-O-rutinoside, is a flavanone-7-Oglycoside
which commonly found in lemons [24]. Eriocitrin has been
also docked with RRF with very acceptable binding affinity which
was in the second rank after hesperidin (Table 1). Similar to its
congener, hesperidin, eriocitrin docked with RRF through hydrogen
binding and hydrophobic interactions (Figure 2). The antibacterial
effects of eriocitrin against gram-positive and gram-negative bacteria
have been reported previously [21,25]

Apigenin, 4′, 5, 7-trihydroxyflavone, is a flavonoid found in
many plants and citrus species [28,29]. Apigenin also found to be
useful as pharmaceutical agents which possess anti-inflammatory
and antioxidative, antitumor, and antibacterial activities [30]. In the
present study, apigenin has considerable binding affinity to dock with
RRF.

More recently, α-carotene, β-carotene and β-cryptoxanthin have
been characterized in peel and pulp extracts of Citrus which inhibited
the growth of both gram-positive and gram-negative bacteria [31].
Our results also showed that β-carotene and β-cryptoxanthin and α-carotene have been docked with RRF with considerable
binding affinities and may involve in antibacterial effects of CL. The
antibacterial and anti-inflammatory effects of these natural pigments
are not a new subject, although our in Silico findings would be a new
insight in this context [32]. In this line, beta-cryptoxanthin belongs
to the class of carotenoids and it converted to vitamin A (retinol) in
human body and is therefore considered as a pro-vitamin A. it is found
mainly in citrus fruit in both free and esterified forms [33]. Betacryptoxanthin
has been docked with RFF throughout hydrophobic
interactions with considerable binding affinity.

D-limonene is a cyclic terpene found in appreciate amount in
citrus species [34]. The antimicrobial activity of d-limonene has
been subject of many investigations (e.g.,[35]). In the present study,
d-limonene has been docked with RRF via hydrophobic interactions
(Figure 2) with moderate binding affinity (Table 1).

Alpha-pinene is a terpenoid compound ubiquitously distributed
among plants including citrus species [36]. Alpha-pinene is an
anti-inflammatory and broad-spectrum phytobiotic [37]. In this
study, α-pinene and citral showed identical binding affinity against
RRF (Table 1) and these terpenoid compounds only took part in
hydrophobic interactions with RRF (Figure 2a-2k). More specifically,
citral or 3, 7-dimethyl-2, 6-octadienal is terpenoid compound found
in citrus family and various plants [38]. In this study, citral A or
geranial showed acceptable binding affinity with RRF (Table 1). Citral
is known as its lemon flavor and has antibacterial effects [39].

Conclusion

The emergence of antibiotic-resistant bacteria and hard access
to antibiotics in different parts of the world motivated researchers
to seek alternative therapeutics. In this continuum, hydro-alcoholic
extracts prepared of CL peel, as a waste product, showed significant
antibacterial activity against E. coli.In silico findings showed that
among phytochemicals found in peel of CL, hesperidin has more
negative binding affinity to RRF compared to gentamicin and would
be considered as a lead molecule for further attempts to design
phytobiotics.